Importance of Candida infection and fluconazole resistance in women with vaginal discharge syndrome in Namibia

Background Vaginal discharge syndrome (VDS) is a common condition. Clinical management targets sexually transmitted infections (STIs) and bacterial vaginosis (BV); there is limited focus on Candida infection as cause of VDS. Lack of Candida treatment coverage and, if present, antifungal resistance may result in VDS treatment failure. This study aimed to determine the prevalence of Candida infection, antifungal resistance, and coinfections in Namibian women with VDS. Methods A cross-sectional study was performed using 253 vaginal swabs from women with VDS in Namibia. Demographic data was collected, and phenotypic and molecular detection of Candida species was performed followed by fluconazole susceptibility testing of Candida isolates. BV was diagnosed using Nugent score microscopy; molecular detection of Chlamydia trachomatis, Neisseria gonorrhoeae and Trichomonas vaginalis was performed. Results Candida species was detected in 110/253 women (43%). Ninety women (36%) had Candida albicans and 24 (9.5%) had non-albicans Candida species. The non-albicans species detected were 19 (17%) Candida glabrata, 4.0 (3.5%) Candida krusei, and 1.0 (0.9%) Candida parapsilosis. Candida albicans were more frequently isolated in younger (p = 0.004) and pregnant women (p = 0.04) compared to non-albicans Candida species. Almost all (98%) Candida albicans isolates were susceptible to fluconazole while all non-albicans Candida species were fluconazole resistant. STIs were diagnosed in 92 women (36%): 30 (12%) with C. trachomatis, 11 (4.3%) N. gonorrhoeae, and 70 (28%) T. vaginalis; 98 (39%) women had BV. Candida infection alone was diagnosed in 30 women (12%), combined with STIs in 42 women (17%) and was concurrent with BV in 38 women (15%). Candida infection was more often detected in swabs from women without C. trachomatis detected (6.4% vs. 16%; OR 0.30; 95% CI 0.10–0.77, p = 0.006). Conclusions The high prevalence of Candida infection, especially those due to non-albicans Candida species that are resistant to fluconazole, is a great concern in our setting and may lead to poor treatment outcomes. Access to microbiological testing for Candida species in the context of syndromic management is warranted.


Background
Vaginal discharge syndrome (VDS) is the most common gynaecological condition among women of reproductive age [1]. Vulvovaginal candidiasis (VVC) is the most common aetiology of VDS, accounting for about 90% of symptomatic vaginal infections [2,3]. Up to 75% of healthy women face symptomatic VVC at least once in Open Access *Correspondence: remco.peters@up.ac.za Dunaiski et al. Antimicrobial Resistance & Infection Control (2022) 11:104 their childbearing years, with some experiencing intermittent and often obdurate forms of the disease [4,5]. A previous study from Namibia reported that symptomatic VVC was present in 61/335 (18.2%) of women newly diagnosed with human immunodeficiency virus (HIV) that were enrolled for antiretroviral therapy [6]. Additionally, recurrent VVC (RVVC) may occur in 10%-20% of women with symptomatic VVC [7]. In two recent studies, RVVC, defined as four or more symptomatic episodes per year, was estimated to occur in 37,390 females per year in Namibia and in over a million females per year in South Africa [8,9]. There are several factors contributing to RVVC, which include treatment failure, coinfections and antifungal resistance [10]. The aetiology in more than 90% of the VVC cases is Candida albicans [11]. Non-albicans Candida species have emerged as an important aetiology of VVC as its prevalence and antifungal resistance is a mounting problem globally [10,11]. The most significant of these nonalbicans Candida species is Candida glabrata owing to its intrinsic resistance or low susceptibility to azoles [12]. Other than C. glabrata, C. krusei, C. tropicalis, C. parapsilosis and, very rarely, Saccharomyces cerevisiae, are other potential pathogens that may lead to VVC [13].
In addition to VVC, VDS may also be caused by sexually transmitted infections (STIs), such as Chlamydia trachomatis, Trichomonas vaginalis, Neisseria gonorrhoeae, and bacterial vaginosis (BV) [2,3]. In Namibia, like other low-to middle-income countries (LMICs), syndromic management of VDS is the standard of care, where clinicians treat patients empirically, without aetiological diagnosis, based on a set of symptoms [14]. This approach is associated with under-treatment as STIs frequently remain asymptomatic, but over-treatment with unnecessary use of antibiotics also occurs [15,16]. Determination of the microbiological aetiology of VDS is essential to guide empirical treatment algorithms and to guide effective prescription of antifungal drugs for presumed candidiasis. Most of such studies focus on STIs and do not include Candida species, or antifungal resistance [15,17,18]. Comprehensive evaluation of microbial aetiology of VDS is essential to better understand the occurrence of Candida species, as well as the intersection with STIs and BV. The aim of this study was to determine the prevalence of Candida species, fluconazole resistance and coinfections in swabs collected from women with VDS in Namibia.

Study design, setting and population
This cross-sectional study was conducted using 253 vaginal swabs collected from women with VDS between February and July 2021 at primary healthcare facilities across Namibia. The vaginal swabs were collected by healthcare workers, which were sent to the diagnostic laboratory for routine diagnostic testing.
The laboratory requisition forms were assessed for inclusion and exclusion in the study. Vaginal swabs from women aged 18 to 49 years of age with 'vaginal discharge' as diagnostic indication recorded by the clinician were included, while swabs were excluded if more than two weeks old.

Detection and identification of Candida isolates
Vaginal swabs were inoculated on the chromogenic Candida agar plates (CHROMagar ™ , France), which were incubated (Thermo Scientific, USA) at 37 °C for 24-36 h. Colonies isolated on chromogenic Candida agar were used to identify Candida spp. according to colony colour, as per manufacturer's instructions. In each test, the reference strain C. albicans American Type Culture Collection (ATCC) 14053 was used for quality control. If there were no visible colonies within 3 days, the sample was considered negative for Candida. Colonies were inoculated on the Sabouraud Dextrose Agar (SDA) plates (Oxoid, United Kingdom) in order to be stored with 50% glycerol (Merck, Germany) at − 20 °C prior to DNA extraction.

Molecular confirmation of Candida species
Deoxyribonucleic acid (DNA) extraction and purification from vaginal swabs were performed using the Quick-DNA ™ Fungal/ Bacterial Miniprep Kit (Zymo Research, USA) according to the manufacturer's guidelines. Multiplex PCR was performed using the One Taq ® Quick-Load 2 × Master Mix (New England BioLabs, USA), a universal Candida primer pair targeting ITS1 and ITS2 and C. albicans specific primers according to Rad et al. [19]. The PCR products were then analysed with the 50 bp DNA ladder (New England BioLabs, USA) by gel electrophoresis through a 2% agarose gel and ultraviolet visualisation [19]. Candida albicans ATCC 14053, C. glabrata CBS2175, C. parapsilosis CBS2195, C. tropicalis CBS94, and C. krusei CBS473 were included in each PCR reaction as positive controls; nuclease-free water (Bio-Concept, Switzerland) was used as negative control.

Fluconazole antifungal susceptibility patterns of Candida isolates
The fluconazole susceptibility of Candida isolates was determined using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) microbroth dilution (MBD) method to determine the minimum inhibitory concentration (MIC) [20]. The EUCAST breakpoints were used to assign the Candida species into the clinical categories "susceptible", "intermediate" and "resistant" [20]. Quality control isolates included C. parapsilosis ATCC 22019 (susceptible) and C. krusei ATCC 6258 (resistant) [21].

Detection of bacterial vaginosis
A Gram-stained vaginal smear prepared from vaginal swab was examined under a microscope and evaluated for BV by Nugent scoring [22,23]. Nugent scores from 0 to 3 are considered as "Normal"; 4 to 6 as "Intermediate"; and 7 to 10 as "BV" [24].

Detection of sexually transmitted infections
Molecular detection of Chlamydia trachomatis and N. gonorrhoeae was performed using the LightMix 480 HT CT/NG assay (TIB MOLBIOL, Berlin, Germany), while a validated in-house real time-PCR assay, as described elsewhere, was performed for detection of T. vaginalis [25].

Statistical analyses
Data were captured into a study-specific Epi Info ™ database version 7.2.4.0 (Centres for Disease Control and Prevention (CDC), USA) and exported into RStudio version 2021.09.1 (RStudio, USA) for analysis. Data are presented as absolute value with proportion, and median with range. The chi-squared test, with Fisher's Exact if appropriate, was used to compare dichotomous variables between groups, while the Mann-Whitney test was used for continuous variables between groups. Logistic regression was used to calculate associations of age and pregnancy between Candida albicans and non-albicans Candida species isolated from women with yeast infections. A p value < 0.05 was considered statistically significant.
There were significant differences between women with C. albicans and non-albicans Candida spp. with regards to age (p = 0.01) and pregnancy (p = 0.002) ( Table 3). Similar results were observed with the multivariate analysis, where both age (p = 0.004) and pregnancy (p = 0.04) were found to be independently associated with C. albicans isolated from vaginal swabs. Candida albicans are more likely to occur in pregnant women (adjusted odds ratio 1.9; 95% CI 1.0-3.5, p = 0.002) and in younger women, as C. albicans is less likely to occur with increasing age (adjusted odds ratio 0.94; 95% CI 0.90-0.98, p = 0.01).

Fluconazole susceptibility pattern
The overall drug susceptibility pattern of Candida isolates against fluconazole is shown in Table 4. Fluconazole resistance was low in C. albicans isolates, but high in all non-albicans Candida isolates: C. glabrata (74%), C. krusei (100%) and C. parapsilosis (100%). There was no significant association between fluconazole susceptibility of C. albicans with demographic factors or coinfections.

Relationship between Candida species, STIs and BV
Multiple infections were common: two or more concurrent infections were detected in 86 (34%) women (Fig. 1).

Discussion
Vaginal discharge syndrome (VDS) is a common cause of gynaecological visits among women in sub-Saharan Africa [1,26,27]. Most studies report only on specific microbiological aetiology of this condition, usually STIs, but only few have studied microbial aetiology comprehensively. Consequently, VVC and antifungal resistance is often not reported [1,15,17,28]. This study provides a comprehensive analysis of microbial aetiology of VDS in Namibian women; it highlights a high prevalence of Candida species, including fluconazole-resistant nonalbicans Candida species, and concurrence with STIs and BV.
In this cross-sectional study, no significant association with older age, pregnancy or HIV seropositive status was observed although these are known risk factors for VVC [39][40][41][42][43]. However, we did observe a significant association between the presence of Candida infection and detection of Chlamydia trachomatis, but not with the other STIs, where C. trachomatis was less likely to be detected in women with Candida (COR, 0.30, p = 0.006). A study by Kruppa and colleagues demonstrated a novel interaction between Chlamydia trachomatis and C. albicans via the binding of elementary bodies of Chlamydia trachomatis to C. albicans yeast and hyphal forms. This binding was shown to considerably decrease the capacity of Chlamydia trachomatis to infect human cervical epithelial cells, thereby decreasing its disease progression [44]. In contrast, another study illustrated that biofilms related to VVC may act as a reservoir for Chlamydia trachomatis [3]. STIs have been suggested in other studies as risk factor for VVC; however, this relationship should be further confirmed [45,46]. In our study, like most other studies looking at the species distribution of Candida, the most prevalent Candida species isolated was C. albicans, followed by C. glabrata [47,48]. Candida glabrata is the most relevant nonalbicans Candida species, owing to its ability to develop acquired resistance subsequent to exposure to azole antifungals [11,12,49]. In our study, low rate of fluconazole resistance was found in C. albicans isolates (< 5%), but most non-albicans Candida isolates (n = 19, 17%) were fluconazole resistant. These findings are similar to reports from Africa and other parts of the world [33,48]. Antifungal resistance of Candida spp. is a mounting problem universally [49][50][51]. High rates of fluconazole resistance has been demonstrated in several countries including China [52], Iran [53], Ethiopia [39], Peshawar [54], Brazil [55], Cameroon [33] and Uganda [48], to mention but a few. The use of azole antifungals may stimulate the selection of resistant subpopulations of Candida by shifting colonisation to more intrinsically resistant species, especially C. krusei or C. glabrata [56]. In our study, nonalbicans species that were not susceptible to fluconazole were detected in 24/253 (9.5%) women with VDS, which is 22% of all women with Candida infection, highlighting the challenge in management of Candida species in the syndromic management context. Since pregnancy predisposes women to VVC, which in turn could increase the risk for poor pregnancy outcomes, it is reassuring that the pregnant women in this study all had C. albicans infection, and not fluconazole-resistant non-albicans Candida, and could therefore be adequately treated [57]. We observed an association between older age and the isolation of non-albicans species vs. C. albicans [4]. Similarly, some studies show that non-albicans species, such C. glabrata, are associated with older age when compared to C. albicans, which may be due to the exposure of several risk factors such as the use of hormonal contraceptives and broad spectrum antifungals [58,59].
Our study demonstrates the complex microbial aetiology of VDS. Several women in this study experienced more than one infection, and some up to three infections at once, which may lead to overlapping diagnosis and conditions. Hence, these findings questions whether empirical approach for the management of VDS based on symptoms is appropriate or not [2].
This study has several limitations. First, limited demographic and clinical information was available from study participants due to collecting specimens submitted to the laboratory. Reliance on the information provided by the requesting clinician might have resulted in some misclassification. Second, the culture and molecular methods used target Candida species that cause vaginal infections; other Candida species might have been missed by these assays.

Conclusion
This study highlights a high prevalence of VVC in women with VDS in Namibia. The high frequency of non-albicans Candida species that are resistant to fluconazole is a great concern and may contribute to poor treatment outcomes. Access to microbiological testing for Candida species in the context of syndromic management is warranted.

Acknowledgements
The author hereby acknowledges the Department of Microbiology, Namibia Institute of Pathology for providing specimens and data required to perform study. In addition, the author also recognizes the Department of Health and Applied Sciences, Faculty of Health and Applied Sciences, Namibia University of Science and Technology, and the Department of Medical Microbiology, Faculty of Health Sciences, University of Pretoria for providing the facilities and support required to perform the experimental work.

Author contributions
CD was responsible for the design of the study, acquisition of data, conducting the experimental work, analysis of data, writing the article and final approval of manuscript. RP, MK and HJ were responsible for supervising the design and implementation of the study, substantively revising the article and approving the submitted version. All authors read and approved the final manuscript.

Funding
CMD has received funding from the University of Pretoria Doctoral Commonwealth Scholarship (20200846). MMK, HJ and RPP did not receive funding for preparation of this manuscript.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.